The overall goals of this protocol are to isolate cardiac mesenchymal stromal cells from arrhythmogenic cardiomyopathy patients, test their adipogenic differentiation aptitude and phenotype their surface markers. This method can help answer key questions in the arrhythmogenic cardiomyopathy field, such as the involvement of cardiac stromal cells in adipo-substitution and underline molecular mechanisms. The main advantage of this technique is that the isolation of stoma cells could be performed with few simple passages from very small heart specimens.
Though this method can provide insight into arrhythmogenic cardiomyopathy study. It can also be applied to other systems such as the study of cardiovascular diseases for which an endomyocardial biopsy is indicated. We first studied the idea for this method when we understood that we could scale down the procedure of stoma cell isolation to apply it to cardiac biopsies and the electrophysiology it could provide to us.
For this protocol, collect an endomyocardial biopsy from the right ventricle of an ACM patient using integrated electroanatomical mapping and cardiac fluoroscopy. Take about five grams of tissue and ship it to the lab in TMES medium. At the lab, set up the biological safety cabinet as needed to carry out the procedure up to the ensomatic digestion.
Then transfer the biopsy to a plate and wash it twice with PBS. At this point, the biopsy may be photo documented under a microscope. Next transfer the biopsy into a two milliliter tube containing one milliliter of the collagenase solution.
Therein, cut the sample into small fragments, between 0.5 and one millimeter wide. Then incubate the tissue for 90 minutes at 37 degrees Celsius with continuous rotating agitation. Next, centrifuge the digested solution, remove the supernatant, and re-suspend the pellet in one milliliter of PBS.
Then centrifuge the tube again, remove the supernatant, and re-suspend the pellet in one milliliter of TMES. Now plate the suspension on a 60 millimeter plate and fill the plate up to three milliliters using TMES, but no further. Then culture and expand the cells according to the text protocol.
Before starting, get the cell dissociation reagent, the washing buffer, and the required antibodies ready to use. When the estimated cell number of the culture reaches three million, wash the 100 millimeter plate twice with 10 milliliters of sterile PBS. Next add five milliliters of a cell dissociation reagent to the cells and incubate the plate for seven to 10 minutes at room temperature to detach the cells.
Once the cells are detached, add 15 milliliters of PBS to the cells and transfer the suspension to a 50 milliliter tube. Now, centrifuge the cells and re-suspend the pellet in one milliliter of washing buffer. Then count the cells and transfer three million to a new tube in a final volume of 1.5 milliliters of washing buffer.
Next, split the suspension into 12 fax tubes. Into each tube, add antibodies used to characterize CMSCs at the recommended concentrations. Be sure to include the isotype controls.
Once prepared, incubate the tubes for 15 minutes at room temperature, shielded from light. Later add one milliliter of wash buffer to each tube to stop the reactions. Then centrifuge the cells, remove the supernatant, and re-suspend the pellets in 250 microliters of washing buffer.
Now proceed to characterize the cells using flow cytometry. After adipogenic differentiation of the cells, use ORO staining to test for lipid accumulation. Before starting, be sure to have ORO working solution.
Prior to staining the cells, remove the medium and wash the cells twice with two milliliters of PBS. Then cover the cells in 4%paraformaldehyde and let them fix for five minutes at room temperature. Next discard the paraformaldehyde and wash the cells twice with two milliliters of PBS.
After the second wash add enough ORO working solution to cover the cells. Then incubate the plate for an hour at room temperature. This incubation must be carried out in an environment where the autosolution will not operate, so full rooms are not recommended.
After the incubation, aspirate the ORO solution and wash the cells with two milliliters of PBS several times until the stain cannot be seen in the PBS wash and until, when viewed under the microscope, non-specific staining is not detected. Now, using an inverted tissue culture phase contrast microscope, capture 20 images of each well at 20x magnification. Details are provided in the text protocol.
CMSCs were isolated from endomyocardial biopsies as described. To confirm their mesenchymal lineage, cells were incubated with appropriate florofour conjugated antibodies and analyzed by flow cytometry. The CMSCs were positive for the specific mesenchymal surface antigens CD29, CD44, and CD105.
The fraction of CD90 positive cells is known to be variable. The markers for endothelial heritage, monocyte or macrophage heritage, onomatopoetic heritage, and for the major histo-compatibility complex were not expressed. Next, the CMSCs were cultured in T.ADIPO medium for adipogenic differentiation and stained with ORO to look for intracellular lipid droplets.
CMSCs from patients with arrhythmogenic cardiomyopathy accumulated more lipid droplets then cells from healthy controls. These differences were measured under differentiation conditions at 72 hours and at seven days. Once mastered, the cell resolution can be done in three hours, the staining can be done in two hours, and the fax analysis can be completed in three hours.
Following this procedure, other methods like drug testing could be performed in order to answer additional questions like can we use stoma cells from the same patients respond to different stimuli. After its development, this technique has paved the way to researchers in the field of arrhythmogenic cardiomyopathy to explore the involvement of human cardiac stroma cells in disease pathogenesis.
